Genetic dissection of the fatty liver QTL Fl1sa by using congenic … · 2017. 8. 26. · may be...

RESEARCH ARTICLE Open Access

Genetic dissection of the fatty liverQTL Fl1sa by using congenic miceand identification of candidate genesin the liver and epididymal fatMiyako Suzuki1, Misato Kobayashi1*, Tamio Ohno2, Shinsaku Kanamori1, Soushi Tateishi1, Atsushi Murai1

and Fumihiko Horio1

Abstract

Background: Nonalcoholic fatty liver disease (NAFLD) is a multifactorial disease caused by interactions betweenenvironmental and genetic factors. The SMXA-5 mouse is a high-fat diet-induced fatty liver model established fromSM/J and A/J strains. We have previously identified Fl1sa, a quantitative trait locus (QTL) for fatty liver on chromosome12 (centromere-53.06 Mb) of SMXA-5 mice. However, the chromosomal region containing Fl1sa was too broad. Theaim of this study was to narrow the Fl1sa region by genetic dissection using novel congenic mice and to identifycandidate genes within the narrowed Fl1sa region.

Results: We established two congenic strains, R2 and R3, from parental A/J-12SM and A/J strains. R2 and R3 strains havegenomic intervals of centromere-29.20 Mb and 29.20–46.75 Mb of chromosome 12 derived from SM/J, respectively. Livertriglyceride content in R2 and R3 mice was significantly lower than that in A/J mice fed with a high-fat diet for 7 weeks.This result suggests that at least one of the genes responsible for fatty liver exists within the two chromosomal regionscentromere-29.20 Mb (R2) and 29.20–46.75 Mb (R3). We found that liver triglyceride accumulation is inversely correlatedwith epididymal fat weight among the parental and congenic strains. Therefore, the ectopic fat accumulation in the livermay be due to organ-organ interactions between the liver and epididymal fat. To identify candidate genes in Fl1sa, weperformed a DNA microarray analysis using the liver and epididymal fat in A/J and A/J-12SM mice fed with a high-fat dietfor 7 weeks. In epididymal fat, mRNA levels of Zfp125 (in R2) and Nrcam (in R3) were significantly different in A/J-12SM

mice from those in A/J mice. In the liver, mRNA levels of Iah1 (in R2) and Rrm2 (in R2) were significantly different in A/J-12SM mice from those in A/J mice.

Conclusions: In this study, using congenic mice analysis, we narrowed the chromosomal region containing Fl1sa totwo regions of mouse chromosome 12. We then identified 4 candidate genes in Fl1sa: Iah1 and Rrm2 from the liverand Zfp125 and Nrcam from epididymal fat.

Keywords: Fatty liver, Congenic, fl1sa, Epididymal fat, Interaction, QTL, Candidate gene, Mouse

* Correspondence: [email protected] of Applied Molecular Biosciences, Graduate School ofBioagricultural Sciences, Nagoya University, Nagoya 464-8601, JapanFull list of author information is available at the end of the article

© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Suzuki et al. BMC Genetics (2016) 17:145 DOI 10.1186/s12863-016-0453-7

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BackgroundNonalcoholic fatty liver disease (NAFLD) is a multifactorialdisease caused by interactions between environmental andgenetic factors. NAFLD is frequently complicated by type 2diabetes, obesity, insulin resistance, and hyperlipidemia. Indeveloped countries, the prevalence of NAFLD reachedapproximately 30% of adults [1, 2]. Environmental fac-tors such as high-fat diets, methionine/choline-deficientdiets, and low-carbohydrate (ketogenic) diets can inducedevelopment of NAFLD [3, 4]. In humans, APOC3variants, PLIN1 mutations, and PNPLA3 variants havebeen reported as genetic factors of NAFLD [1, 5].However, the underlying mechanism of how environ-mental and genetic factors interact to cause NAFLD islargely unknown.SMXA-5 mouse is one of the SMXA recombinant in-

bred (RI) strains established from parental SM/J and A/Jstrains [6]. We have previously found that SMXA-5 micedeveloped severe fatty liver on a high-fat diet, althoughparental SM/J and A/J mice were resistant to fatty liver[7]. To elucidate the genetic mechanism of NAFLD inSMXA-5 mice, we performed a quantitative trait locus(QTL) analysis using (SM/J × SMXA-5)F2 intercrossedmice and identified a significant QTL (Fl1sa, fatty liver 1in SMXA RI strains) for liver weight, liver triglyceride, andliver total lipid content on centromere-53.06 Mb of mousechromosome 12 [8]. This QTL for the development offatty liver was attributed to the A/J allele. To confirm theeffect of Fl1sa, we analyzed the fatty liver phenotypes inA/J-12SM chromosomal substitution (consomic) mice, inwhich chromosome 12 of the SM/J mouse was introduced

into the A/J mouse genome. Consequently, we demon-strated that liver total lipid, liver triglyceride, and liverweight in A/J-12SM mice were significantly lower thanthose in A/J mice on a high-fat diet for 7 weeks, but notlower than those in mice on the normal diet [9]. We veri-fied the effect of the A/J-derived Fl1sa on the develop-ment of the high-fat diet-induced fatty liver. We alsoidentified three candidate genes, Iah1, Rrm2, Prkd1, inFl1sa by DNA microarray analysis using livers of A/J andA/J-12SM mice fed on a high-fat diet.In this study, to narrow the chromosomal region of

Fl1sa, first, we constructed two strains of congenic mice,R2 and R3, from parental A/J and A/J-12SM strains (Fig. 1),and then analyzed the lipid accumulation in their liver. Asthe liver triglyceride content in R2 and R3 congenic micewas significantly lower than that in A/J mice, the Fl1saregion was narrowed to two parts of chromosome 12.Subsequently, in both the narrowed Fl1sa regions, weattempted to identify candidate genes in Fl1sa using DNAmicroarray analyses of liver and epididymal fat from A/Jand A/J-12SM mice. As a result, we identified several can-didate genes in both the liver and epididymal fat.

MethodsAnimalsThe A/J strain was purchased from Japan SLC (Hamama-tsu, Japan) and maintained in our facility at the GraduateSchool of Bioagricultural Sciences, Nagoya University. TheA/J-12SM strain (chromosome 12 consomic mouse) wasconstructed from the A/J (recipient) and SM/J strain(donor) at the Institute for Laboratory Animal Research

Fig. 1 Chromosome 12 constructs of consomic and congenic strains. The genomes of consomic and congenic strains consist of recipient A/J anddonor SM/J genomes. White and black boxes represent A/J and SM/J genomic intervals, respectively. Gray boxes represent unclear regions wherethe genomic intervals were derived from the A/J or SM/J strains. Arrow represents the Fl1sa region (centromere-53.06 Mb) on chromosome 12

Suzuki et al. BMC Genetics (2016) 17:145 Page 2 of 10

(Nagoya University) as previously described [10]. The R2and R3 congenic strains were constructed from A/J (recipi-ent) and A/J-12SM strains (Fig. 1). Practically, to produceN1 mice that were heterozygous for the SM/J-derivedchromosome 12 on the background strain A/J, male A/J-12SM mice were mated with female A/J mice. The N1 micewere then backcrossed to A/J mice to produce heterozy-gous mice carrying novel genomic intervals of interest onchromosome 12. Heterozygous mice were mated with A/Jmice and their progeny with the same genomic intervalswere obtained. Finally, to generate mice carryinghomozygous novel genomic intervals, the heterozygousmice were intercrossed. Mouse tails were collected underanesthesia induced using isoflurane. Genomic DNA was ex-tracted from tails using the DNeasy Blood & Tissue kit(QIAGEN). Microsatellite markers and single nucleotide-polymorphisms (SNPs) were used for genotyping of R2 andR3 genomic DNA (Additional file 1). The physical positionsof microsatellite markers and SNPs were taken from theEnsembl database (GRCm38.P4). We previously mappedthe QTL (Fl1sa) for fatty liver by using male (SM/JxSMXA-5)F2 mice [8]. Subsequently, we have confirmed that theFl1sa contributed to fatty liver traits in male A/J-12SM con-somic mice [9]. Therefore, in this study, all procedures wereperformed by using only male mice. All mice were main-tained in our facilities at a temperature of 23 ± 2 °C, humid-ity of 55 ± 5%, and a light/dark cycle of 12 h. Mice wereweaned at 3 weeks of age and housed at one animal percage. All mice had access to food and tap water ad libitum.

Experimental schedule and diet compositionMale A/J, A/J-12SM, R2, and R3 mice were fed CE-2 stand-ard chow (CLEA Japan, Inc., Japan) until 6 weeks of age,thereafter fed a high-fat diet (D07053003; Research Diets,New Brunswick, NJ, USA) from 6 to 13 weeks of age. Thecomposition (per kg diet) of the high-fat diet was as follows:casein, 209 g; carbohydrate (corn starch: sucrose: maltodex-trin 10, 94:100:175), 369 g; mineral mix S10022G, 35 g; vita-min mix V10037, 10 g; choline bitartrate, 2 g; corn oil, 35 g;lard, 300 g; and cellulose BW200, 40 g. The content of fatin this diet was 56% (energy %). The body weight and foodintake were measured every week during the experimentalperiod (6–13 weeks of age). At 13 weeks of age, all micewere sacrificed by cervical dislocation after 4-h fast (9:00–13:00 h). The liver and white adipose tissue (subcutaneousfat, epididymal fat, mesenteric fat, and retroperitoneal fat)were harvested, weighed, and immediately frozen using li-quid nitrogen. Blood samples were collected from orbitalveins to measure serum lipids. All procedures and animalcare were approved by the Animal Experiment Committee,Graduate School of Bioagricultural Sciences, NagoyaUniversity (approval No. 2013021803, 2014020403,2015022603) and were carried out in accordance with theRegulations on Animal Experiments of Nagoya University.

Measurement of serum triglyceride and cholesterolSerum triglyceride and cholesterol concentrations weremeasured using the Triglyceride-E test Kit (Wako PureChemical Industries, Japan) and the Cholesterol-E test Kit(Wako Pure Chemical Industries, Japan), respectively.

Hepatic triglyceride and total lipid content analysisFrozen livers (approximately 0.5 g each) were homoge-nized using chloroform-methanol (2:1), and statically ex-tracted overnight. A portion of the organic extract wasdried, and the hepatic triglyceride content was measuredusing the Triglyceride-E test Kit. The remaining organicsolvent was used for total liver lipid measurement aspreviously described by Folch et al. [11].

DNA microarray analysis in epididymal fatTotal RNA was isolated using the TRI reagent (MolecularResearch Center Inc.) and RNeasy Mini Kit (QIAGEN)from frozen epididymal fat obtained from 4-h fasted A/Jand A/J-12SM male mice that were fed the high-fat diet for7 weeks. Total RNA from three mice per strain was pooledfor each chip. Whole transcripts from epididymal fats weremeasured using a Mouse Genome ST 2.0 array (Affyme-trix). Raw data were normalized with RMA-sketch algo-rithm by Affymetrix Expression Console Software ver.1.3.0.The microarray data have been deposited in the NCBIGene Expression Omnibus (GEO) (GSE79281). The detailsof expression profiles are shown in Additional file 2.

Real-time RT-PCRTotal RNA was isolated using the TRI reagent from fro-zen liver and epididymal fat of A/J, A/J-12SM, R2, andR3 male mice that were fed the high-fat diet for 7 weeks.Isolated RNA was then treated with TURBO DNA-freekit (Ambion) to eliminate DNA contamination. There-after, the cDNA was synthesized from DNase-treatedtotal RNA using the High Capacity Reverse Transcrip-tion kit (Applied Biosystems). Gene expression was de-termined using the StepOnePlusTM Real-Time PCRSystem (Applied Biosystems) with the ThunderbirdqPCR Mix or the Thunderbird SYBR qPCR Mix(TOYOBO, Japan). Each mRNA level was normalized tothe corresponding β-actin mRNA level. To determinethe mRNA level of Iah1, we used TaqMan probes (Taq-Man Gene Expression Assays, Mm00509467_m1;Applied Biosystems). The details of primers used for theSYBR Green assays are shown in Additional file 3.

Statistical analysisAll results were expressed as mean ± SEM. One-wayANOVA and subsequent Dunnett’s test were used to com-pare the means of A/J-12SM, R2, and R3 with those of A/Jmice. Student’s t-test was used to compare the means be-tween A/J and A/J-12SM mice. The correlation between


fatty liver parameters (liver weight and liver triglyceridecontent) and epididymal fat weight were analyzed usingSpearman correlation analysis. Differences with p < 0.05were regarded as significant. Statistical analyses wereperformed by using StatView 5.0 software (SAS Institute,Cary, NC).

ResultsPhenotypic analysis in A/J, A/J-12SM, R2, and R3 mice thatwere fed the high-fat diet for 7 weeksAlthough initial body weight and food intake were not dif-ferent in each strain, the final body weight in A/J-12SM

and R3 mice was significantly lower than that in A/J mice(Table 1). Liver and mesenteric fat weights in A/J-12SM

mice were significantly lower than those in A/J mice. Onthe other hand, epididymal fat and retroperitoneal fatweight in A/J-12SM mice were significantly higher thanthose in A/J mice. R2 and R3 mice did not show any sig-nificant differences in tissue weight compared to A/J mice.However, liver weight in R2 and R3 mice tended to beslightly lower than that in A/J mice. In addition, epididy-mal fat weight in R2 mice tended to be higher than that inA/J mice. Liver triglyceride content was significantly lowerin A/J-12SM, R2, and R3 mice, compared with that in A/Jmice (Fig. 2a). The changes in liver total lipid content inall strains were similar to those in liver triglyceride con-tent (Fig. 2b). Liver triglyceride and liver total lipid in R2and R3 mice showed intermediate values between those ofA/J and A/J-12SM mice. These results suggest that thegenes responsible for fatty liver exist in the centromere-29.20 Mb (SM/J region in R2 strain) and 29.20–46.75 Mb(SM/J region in R3 strain) regions of chromosome 12, re-spectively (Fig. 1). Serum triglyceride concentration did

not differ among all strains. Serum total cholesterol con-centration in A/J-12SM mice was significantly lower thanthat in A/J mice; however, there were no differences in R2and R3 mice relative to the A/J mice (Fig. 2c and d).

DNA microarray analysis of epididymal fat in A/J miceand A/J-12SM miceLiver triglyceride and total lipid in A/J-12SM mice weresignificantly lower than those in A/J mice (Fig. 2a and b).An inverse correlation in tissue weight was observed be-tween epididymal fat and the liver (r = −0.701, p < 0.0001,Fig. 3a). In addition, there was an inverse correlation be-tween epididymal fat weight and liver triglyceride (r =−0.539, p < 0.0001, Fig. 3b). These results suggest that epi-didymal fat is an important tissue for the development offatty liver. We then performed DNA microarray analysisin A/J and A/J-12SM mice using total RNA obtained fromepididymal fat. We identified genes whose expressionlevels were changed 1.68-fold in A/J-12SM

mice compared to those in A/J mice (Additional file 2 andTable 2). In epididymal fat, 19 genes were differentiallyexpressed between the A/J and A/J-12SM mice in thecentromere-46.75 Mb region on chromosome 12. Thegenes Pfn4, Fkbp1b, Apob, Nt5c1b, Ntsr2, Zfp125, Rsad2,Cmpk2, and Allc were found in the R2 interval (centro-mere-29.20 Mb) on chromosome 12 (Table 2). Further-more, the genes Sh3yl1, Slc26a3, Gdap10, Cdhr3, Efcab10,Mir680-3, Dgkb, Nrcam, Stxbp6, and Nova1 were found inthe R3 interval (29.20–46.75 Mb) on chromosome 12. Inorder to validate the expression of these genes, we per-formed real-time RT-PCR (except for Mir680-3, because itis a micro-RNA). We confirmed significant differences ingene expression levels of 6 genes (Ntsr2, Zfp125, Gdap10,

Table 1 Body weight, food intake, and tissue weight in A/J, A/J-12SM consomic mice and R2 and R3 congenic mice fed the high-fat diet

A/J A/J-12SM R2 R3

Body weight (g)

0 weeks of feeding with the HFD (Initial) 21.9 ± 0.3 22.1 ± 0.3 21.4 ± 0.4 21.2 ± 0.5

7 weeks of feeding with the HFD (Final) 39.4 ± 0.4 35.8 ± 0.7** 38.2 ± 0.9 35.6 ± 0.7**

Food intake (g/g BW/day)a

3 weeks of feeding with the HFD 0.065 ± 0.003 0.065 ± 0.001 0.065 ± 0.002 0.071 ± 0.003

6 weeks of feeding with the HFD 0.053 ± 0.002 0.058 ± 0.001 0.052 ± 0.001 0.056 ± 0.002

Weight of tissues (g/100 g BW)

Liver 3.66 ± 0.07 2.99 ± 0.08** 3.40 ± 0.07 3.47 ± 0.10

Subcutaneous fatb 3.86 ± 0.12 3.48 ± 0.12 3.73 ± 0.19 3.55 ± 0.17

Epididymal fat 4.82 ± 0.18 6.24 ± 0.21** 5.35 ± 0.16 4.97 ± 0.18

Mesenteric fat 3.60 ± 0.13 2.90 ± 0.11** 3.56 ± 0.17 3.32 ± 0.11

Retroperitoneal fat 1.26 ± 0.06 1.52 ± 0.04** 1.20 ± 0.04 1.40 ± 0.07

Each value is expressed as the mean ± SEMn = 13–16, ** p < 0.01, significant difference versus A/J strain by Dunnett’s testaBW, body weightbSubcutaneous fat was dissected between the root of the forefoot and the hind leg on right side of the body


Nrcam, Stxbp6, and Nova1) between A/J and A/J-12SM

mice (Fig. 4a and b). Thus, we identified 6 candidate genesfrom epididymal fat.

Expression levels of candidate genes in congenic strainsWe previously performed a DNA microarray analysis usingmRNA from the liver and succeeded in identifying

candidate genes Iah1 and Rrm2 in the R2 region (data havebeen deposited in the NCBI GEO (GSE67340)) [9]. Fur-thermore, in this study, we succeeded in identifying candi-date genes Ntsr2, Zfp125, Gdap10, Nrcam, Stxbp6, andNova1, in epididymal fat from A/J and A/J-12SM mice byDNA microarray analysis and real-time RT-PCR (Fig. 4).Subsequently, we analyzed the mRNA levels of candidate

Fig. 2 Liver lipids and serum lipids of A/J, A/J-12SM, and congenic strains. a Liver triglyceride concentration, b Liver total lipid concentration, c Serumtriglyceride concentration, and d Serum total cholesterol concentraion of A/J, A/J-12SM, and congenic strains fed the high-fat diet for 7 weeks (n= 14–16,*p< 0.05, **p< 0.01 versus A/J mice by Dunnett’s test)

Fig. 3 Correlation of fatty liver phenotype with epididymal fat weight. a A scatter plot of liver weight versus epididymal fat weight and b a scatterplot of liver triglyceride versus epididymal fat weight. Data were pooled from all mice fed the high-fat diet for 7 weeks (n = 59, correlation coefficientand p-value were calculated by Spearman correlation analysis)


genes in congenic mice. In the liver, Iah1 (isoamyl acetate-hydrolyzing esterase 1 homolog (Saccharomyces cerevisiae);21.31 Mb in the R2 region) mRNA level was significantlyhigher in A/J-12SM and R2 mice than in A/J mice (Fig. 5a).Rrm2 (Ribonucleotide reductase M2; 24.70 Mb in R2 re-gion) mRNA level was significantly lower in A/J-12SM andR2 mice than in A/J mice. In the epididymal fat, Ntsr2(neurotensin receptor 2; 16.65 Mb in R2 region) mRNAlevels tended to be higher in A/J-12SM and R2 mice than inA/J mice (Fig. 5b). Zfp125 (Zinc finger protein 125;20.89 Mb in R2 region) mRNA levels were significantlylower in A/J-12SM and R2 mice than in A/J mice. Gdap10(ganglioside-induced differentiation-associated-protein 10;32.82 Mb in R3 region), Stxbp6 (syntaxin binding protein 6(amisyn); 44.85 Mb in R3 region), and Nova1 (neuro-onco-logical ventral antigen 1; 46.69 Mb in R3 region) mRNAlevels were significantly different in A/J-12SM mice, but notin R3 mice compared to those in A/J mice (Fig. 5c).Nrcam (neuronal cell adhesion molecule; 44.32 Mb in R3region) mRNA levels were significantly lower in A/J-12SM

and R3 mice than in A/J mice. In summary, only Zfp125mRNA levels were significantly different between R2 andA/J mice, and only Nrcam mRNA levels were significantlydifferent between R3 and A/J mice. These results suggestthat Zfp125 and Nrcam in epididymal fat are candidategenes in Fl1sa.

DiscussionWe previously found that SMXA-5 mice developed a fattyliver induced by a high-fat diet, and further identifiedFl1sa in the region centromere-53.06 Mb on chromosome12 as the fatty liver QTL. We then determined the A/J-de-rived allele of Fl1sa to be the cause of fatty liver. In thisstudy, we chose a congenic mapping strategy to narrowthe Fl1sa region and conducted a microarray analysis toidentify candidate genes in Fl1sa.First, we narrowed the chromosomal region of Fl1sa

because the chromosomal region spanned by Fl1sa was ex-tremely broad (centromere-53.06 Mb, 611 genes). There-fore, we established the R2 and R3 congenic strains (Fig. 1)and then analyzed the liver lipid accumulation of these con-genic mice that were fed the high-fat diet for 7 weeks. Finalbody weight in A/J-12SM and R3 mice was significantlylower than that in A/J mice, even though food intake wasthe same between the R3 congenic mice and the A/J mice(Table 1). Liver triglyceride content in A/J-12SM, R2, andR3 mice was significantly lower than that in A/J mice(Fig. 2a); however, the liver total lipid content in R2 and R3mice was not significantly changed compared to that in A/Jmice (Fig. 2b). The results of liver lipid analysis showed thatR2 and R3 mice had a smaller effect on fatty liver comparedto A/J-12SM mice. Both serum triglyceride and total choles-terol concentrations were not different in R2 and R3 mice

Table 2 Up-regulated and down-regulated genes in centromere-46.75 Mb (R2 and R3 regions) of chromosome 12 in the epididymalfat in the A/J-12SM consomic strain

Symbol Gene name Position (Mbp) Region Fold changea Gene bank ID

Pfn4 profilin family, member 4 4.76 R2 0.525 NM_028376

Fkbp1b FK506 binding protein 1b 4.83 R2 0.557 NM_016863

Apob apolipoprotein B 7.97 R2 7.460 NM_009693

Nt5c1b 5’-nucleotidase, cytosolic IB 10.36 R2 0.296 NM_027588

Ntsr2 neurotensin receptor 2 16.65 R2 1.896 NM_008747

Zfp125 zinc finger protein 125 20.89 R2 0.104 AJ005350

Rsad2 radical S-adenosyl methionine domain containing 2 26.44 R2 0.580 NM_021384

Cmpk2 cytidine monophosphate (UMP-CMP) kinase 2, mitochondrial 26.46 R2 0.463 NM_020557

Allc allantoicase 28.55 R2 0.366 NM_053156

Sh3yl1 Sh3 domain YSC-like 1 30.91 R3 0.565 NM_013709

Slc26a3 solute carrier family 26, member 3 31.39 R3 0.361 ENSMUST00000001254

Gdap10 ganglioside-induced differentiation-associated-protein 10 32.82 R3 1.928 BC052902

Cdhr3 cadherin-related family member 3 33.03 R3 0.557 NM_001024478

Efcab10 EF-hand calcium binding domain 10 33.39 R3 0.578 NM_029152

Mir680-3 microRNA 680-3 35.19 R3 2.545 NR_030449

Dgkb diacylglycerol kinase, beta 37.88 R3 1.879 NM_178681

Nrcam neuronal cell adhesion molecule 44.32 R3 0.529 NM_176930

Stxbp6 syntaxin binding protein 6 (amisyn) 44.85 R3 3.307 NM_144552

Nova1 neuro-oncological ventral antigen 1 46.69 R3 1.908 NM_021361

Up-regulated (log20.75, >1.68-fold) and down-regulated (log2-0.75,

from those in A/J mice (Fig. 2c and d). In both congenicmice, serum lipid levels did not correlate with the accumu-lation of triglycerides in the liver. These results suggest thatat least one of the genes responsible for fatty liver existswithin the chromosomal region of the SM/J allele in R2(centromere-29.20 Mb) and R3 (29.20–46.75 Mb) mice, re-spectively. The genes in the R2 and R3 regions were notfound to be effective in altering the serum lipid concentra-tion; however, genes in the R3 region might affect bodyweight gain. Moreover, genes in these regions mightinteract with each other because liver triglyceride and livertotal lipid content in R2 and R3 mice were not as low asthose in A/J-12SM mice.Second, we attempted to identify candidate genes in

Fl1sa in the R2 and R3 chromosomal region. Wepreviously used DNA microarray to analyze the compre-hensive gene expression of livers in A/J and A/J-12SM

mice that were fed the high-fat diet for 7 weeks [9]. In thisstudy, we refined the list of genes in this region whose

expression levels in the liver were different between A/Jand A/J-12SM mice (1.68-fold) in the R2and R3 regions (centromere-29.20 Mb and 29.20–46.75 Mb, respectively). We explored candidate genes hav-ing cis-acting expression in each chromosomal region ofR2 and R3 mice. Consequently, we succeeded in isolatingtwo genes, Iah1 (21.31 Mb) and Rrm2 (24.70 Mb), as can-didate genes in the R2 region (Fig. 5a). Iah1 was identifiedas an esterase that exhibits hydrolytic activity against acet-ate esters in yeast [12]. We previously reported that mouseIah1 mRNA is broadly expressed in the liver, kidney, epi-didymal fat, lung, spleen, and muscle [9]. Furthermore,stable overexpression of mouse Iah1 in Hepa1-6 cells sup-pressed the mRNA expression of lipid metabolism-relatedgenes Cd36 and Dgat2 [9]. Our data suggested that mouseIah1 prevents the development of fatty liver by suppress-ing Cd36 and Dgat2 gene expression. Thus, we suggestthat high expression of Iah1 contributes to the decreasedliver triglyceride content in R2 congenic mice. Rrm2

Fig. 4 mRNA levels of differentially expressed genes existing in the genomic region of R2 or R3 congenic strains. Genes differentially expressed inepididymal fat between A/J and congenic mice were selected from a DNA microarray analysis (Table 2). The mRNA levels of selected genes exist in thegenomic region of R2 congenic (a) or R3 congenic (b) strains. The mRNA levels of selected genes were measured using real-time RT-PCR. Epididymal fatswere collected from A/J or A/J-12SM mice fed the high-fat diet for 7 weeks (n= 5–6, *p< 0.05, **p< 0.01 versus A/J strain by Student’s t-test)


encodes the β-subunit of the protein ribonucleotide reduc-tase, a rate-limiting enzyme involved in de novo dNTP bio-synthesis [13]. It was reported that RRM2 overexpressionwas observed in various types of cancer [13], and that pa-tients with liver cirrhosis and hepatocellular carcinoma ex-hibited high levels of RRM2 [14]. However, the relationshipbetween Rrm2 and fatty liver has not been clarified. Fromthe present results, we identified Iah1 and Rrm2 as candi-date genes in the liver for the development of fatty liver.On the other hand, liver weight and liver triglyceride con-

tent are inversely correlated with epididymal fat weight(Fig. 3a and b). These results suggest that controlling theepididymal fat weight contributes to triglyceride accumula-tion in the liver. In addition, it was reported that liver lipidaccumulation and epididymal fat weight showed inversecorrelation in other mouse strains [15, 16]. To identify dif-ferentially expressed genes in epididymal fat, we performedDNA microarray analysis using RNAs from epididymal fatof A/J and A/J-12SM mice. We detected 19 genes, whoseexpression levels in epididymal fat were changed betweenA/J and A/J-12SM mice (1.68-fold) withinthe R2 and R3 chromosomal regions (Table 2). Subse-quently, we validated 6 candidate genes whose mRNAlevels in A/J-12SM mice were significantly different fromthose in A/J mice: Ntsr2 (16.65 Mb), Zfp125 (20.89 Mb),Gdap10 (32.82 Mb), Nrcam (44.32 Mb), Stxbp6

(44.85 Mb), and Nova1 (46.69 Mb) (Fig. 4a and b). Finally,we selected Zfp125 (20.89 Mb, in R2 region) and Nrcam(44.32 Mb, in R3 region) as candidate genes in epididymalfat, because their mRNA levels in R2 and R3 congenic micewere significantly different in accordance with the changein A/J-12SM mice compared to A/J mice (Fig. 5b and c).Zfp125 is a zinc finger protein expressed in many tissuesand might be involved in the regulation of cellular pro-cesses such as cell proliferation and transformation [17].Nrcam is a neuronal cell adhesion molecule that mediatesneuron-neuron and neuron-glia adhesion [18]. Further-more, it was reported that Nrcam was induced in the liverof Fisher-344 rats fed 2-aminoanthracene [19]. Further in-vestigation is needed to clarify the functions of these twogenes in lipid metabolism.The R2 and R3 regions on mouse chromosome 12 are

syntenically conserved in three regions on human chromo-somes 2, 7, and 14. In these syntenic regions, human Gen-ome Wide Association Studies (GWAS) for metabolicsyndrome identified 205 SNPs, which were shown in GWAScatalog (NHGRI-EBI Catalog of published genome-wideassociation studies, http://www.ebi.ac.uk/gwas/home). How-ever, there are no SNPs associated with metabolic syndromein 4 candidate genes (Iah1, Rrm2, Zfp125, and Nrcam).Moreover, we previously performed exome analysis in A/

J and SM/J (data were deposited in DDBJ Sequence Read

Fig. 5 mRNA levels of candidate genes in two congenic strains. The mRNA levels of candidate genes in liver (a) and epididymal fat (b and c)were measured using real-time RT-PCR. A/J, A/J-12SM, R2, or R3 mice were fed the high-fat diet for 7 weeks (n = 6–8, *p < 0.05, **p < 0.01 versusA/J mice by Dunnett’s test). Physical positions and the congenic regions of each gene are given in parentheses


http://www.ebi.ac.uk/gwas/home

Archive, Accession No. DRA002145). We identified non-synonymous SNPs between A/J and SM/J mice (48 genes)in the genomic region R2 and R3 from the exome data(Additional files 4 and 5). The genes having non-synonymous SNPs might cause the change in the functionof their translated proteins. We also consider these genes aspotential candidate genes for fl1sa. At present, we cannotassess the importance of amino acid substitutions in eachgene. In Iah1 gene, we confirmed the two non-synonymousSNPs and these SNPs lead to amino acid substitutions(S37A and G75E in A/J strain, Additional files 4 and 5).Other candidate genes identified by our DNA microarrayanalysis did not have the non-synonymous SNPs.

ConclusionsIn this study, by using R2 and R3 congenic mice, we identi-fied Iah1 and Rrm2 from the liver, and Zfp125 and Nrcamfrom epididymal fat as candidate genes in Fl1sa. Althoughthe relationship between these genes and fatty liver has notbeen previously reported, we demonstrated that Iah1 over-expression affected the expression of lipid metabolism-related genes in Hepa1-6 cells [9]. Therefore, at present, weare examining the incidence of fatty liver in Iah1-knockoutmice. We hypothesize that ectopic fat accumulation in theliver was brought by the organ-organ interaction betweenthe liver and epididymal fat. Thus, Zfp125 and Nrcam inepididymal fat might affect liver triglyceride accumulationthrough the regulation of epididymal fat weight. In futureexperiments, to uncover the molecular mechanism under-lying the relation between these candidate genes and lipidmetabolism, we will need to perform overexpression orknockdown experiments using candidate genes in hepato-cytes or adipocytes. Overall, this study is the first step toelucidating the mechanism of fatty liver development coin-ciding with changes in fat distribution.

Additional files

Additional file 1: Sequences of primers used for genotyping. (DOCX 14 kb)

Additional file 2: Expression profiles of all genes in R2 and R3regions. (XLSX 43 kb)

Additional file 3: Sequences of primers used for real-time RT-PCR.(DOCX 21 kb)

Additional file 4: Allelle variants on chromosome 12 (Centromere-46.75 Mb)in A/J strain versus C57BL/6 strain. (XLSX 15 kb)

Additional file 5: Allelle variants on chromosome 12 (Centromere-46.75 Mb)in SM/J strain versus C57BL/6 strain. (XLSX 13 kb)

AbbreviationsAPOC3: Apolipoprotein C-3; Cd36: Cd36 antigen; Dgat2: Diacylglycerol O-acyltransferase 2; Fl1sa: Fatty liver 1 in SMXA RI strains; Gdap10: Ganglioside-induced differentiation-associated-protein 10; Iah1: Isoamyl acetate-hydrolyzing esterase 1 homolog (Saccharomyces cerevisiae);NAFLD: Nonalcoholic fatty liver disease; Nova1: Neuro-oncological ventralantigen 1; Nrcam: Neuronal cell adhesion molecule; Ntsr2: Neurotensinreceptor 2; PLIN1: Perilipin-1; PNPLA3: Patatin-like phospholipase domaincontaining protein 3; Prkd1: Protein kinase D1; QTL: Quantitative trait locus;

RI: Recombinant inbred; Rrm2: Ribonucleotide reductase M2; SOX4: SRY (sexdetermining region Y)-box 4; Stxbp6: Syntaxin binding protein 6 (amisyn);Zfp125: Zinc finger protein 125

AcknowledgementsThis work supported by the program for Leading Graduate Schools“Integrative Graduate Education and Research in Green Natural Sciences”,MEXT, Japan (to MS).

FundingThis work was supported by a Grant-in-Aid for Scientific Research (C) (No.25450166) from the Japan Society for the Promotion of Sciences, a grantfrom the Uehara Memorial Foundation (to MK).

Availability of data and materialsThe dataset supporting the conclusions of this article is available in the GEOrepository [http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79281].

Authors’ contributionsMS performed the experiments and wrote this manuscript. MK and FHcontributed to the design of the experiments, interpretation of the data andediting of this manuscript. TO contributed to interpretation of the data andconstruction of congenic mice. SK contributed to analysis of congenicphenotypes. ST contributed to construction of congenic mice. AM contributedto interpretation of the data. All authors have read and approved the finalmanuscript.

Competing interestsThe authors declare that they have no competing interests.

Consent for publicationNot applicable.

Ethics approval and consent to participateAll procedures and animal care were approved by the Animal ExperimentCommittee, Graduate School of Bioagricultural Sciencies, Nagoya University(approval No.2013021803, 2014020403, 2015022603) and carried out inaccordance with the Regulations on Animal Experiments of NagoyaUniversity.

Author details1Department of Applied Molecular Biosciences, Graduate School ofBioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.2Division of Experimental Animals, Center for Promotion of Medical Researchand Education, Graduate School of Medicine, Nagoya University, Nagoya466-8550, Japan.

Received: 1 August 2016 Accepted: 27 October 2016

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AbstractBackgroundResultsConclusions

BackgroundMethodsAnimalsExperimental schedule and diet compositionMeasurement of serum triglyceride and cholesterolHepatic triglyceride and total lipid content analysisDNA microarray analysis in epididymal fatReal-time RT-PCRStatistical analysis

ResultsPhenotypic analysis in A/J, A/J-12SM, R2, and R3 mice that were fed the high-fat diet for 7 weeksDNA microarray analysis of epididymal fat in A/J mice and A/J-12SM miceExpression levels of candidate genes in congenic strains

DiscussionConclusionsAdditional filesshow [a]AcknowledgementsFundingAvailability of data and materialsAuthors’ contributionsCompeting interestsConsent for publicationEthics approval and consent to participateAuthor detailsReferences

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